1. Anti-Phospholipid Antibody Syndrome The Annexin A5 Competition Assay as a Diagnostic Tool

2. Anti-Phospholipid Antibody Syndrome An acquired hypercoagulable state Affecting young and middle age individuals Associated with Associated with Arterial and venous thrombosis Recurrent fetal loss (Pregnancy vascular complication) Recurrent abortion Recurrent abortion Premature delivery Premature delivery Severe pre-eclampsia Severe pre-eclampsia

3. Anti-PhospholipidAntibody Syndrome (APS) Mechanisms of APS – uncertain Mechanisms of APS – uncertain In vitro, APL-ab exhibit multiple specificity to the phospholipid-protein complexes In vitro, APL-ab exhibit multiple specificity to the phospholipid-protein complexes –  2 - glycoprotein I (anti-  2 - GPI) –Prothrombin –Cardiolipin –Phosphatidylserine –Phosphatidylethanolamine –Phosphatidic acid

4. Anti-Phospholipid Antibody Syndrome Laboratory Diagnosis Demonstration of anti-phospholipid antibodies immune-detection assay: anti-cardiolipin / anti-β 2 GPI immune-detection assay: anti-cardiolipin / anti-β 2 GPI Phospholipid-dependent clotting time: Lupus Anti Coagulant Phospholipid-dependent clotting time: Lupus Anti Coagulant (APTT variant, dRVVT, other) - Prolongation of the clotting time - Correction by excess phospholipids

5. Anti-Phospholipid Antibody Syndrome Difficulties Assays are heterogeneous with Assays are heterogeneous with methodological and practical limitations methodological and practical limitations Correlation with clinical presentation not entirely apparent Correlation with clinical presentation not entirely apparent

6. Anti-Phospholipid Antibody Syndrome Hypothesis Antiphospholipid autoantibodies interfere with the anticoagulant effect of the natural anticoagulant protein Annexin A5 Resulting in accelerated prothrombotic activity

7. β 2 GPI Annexin V APLab Anionic phospholipid Activated platelet Pro- thrombin Thrombin FXa FVa Ca ++

8. Anti-phospholipid Syndrome Effect of Patient Plasma on Binding of Annexin V

9. Competition Between Anti-Phospholilpid Autoantibodies and Annexin V on Platelet Phospholipids

10. Anti-phospholipid Antibody Syndrome Flow Cytometric Determination of Anti-Platelet Phospholipid Autoantibodies Aaron Tomer, J Lab Clin Med 2002;139:147-54

11. Conclusion  Patients’ antibodies interfere with the binding of Annexin V to the platelet anionic-phospholipids  The displacement of annexin V by patients’ antibodies may result in accelerated procoagulant activity, thereby promoting thrombogenesis in APS Aaron Tomer, J Lab Clin Med 2002;139:147-54. Anti-phospholipid Antibody Syndrome

12. Detection of antibody-mediated reduction of annexinA5 anticoagulant activity in plasmas of patients with the antiphospholipid syndrome. Jacob H. Rand, Xiao-Xuan Wu, Robert Lapinski, Waander L. van Heerde, Chris P. Reutelingsperger, Pojen P. Chen, and Thomas L. Ortel. Blood. 2004;104: 2783-2790. Commentary on Rand et al, page 2783 Antiphospholipid antibodies: a mosaic of pathogenic effects? Silvia S. Pierangeli MOREHOUSE SCHOOL OF MEDICINE The elucidation of the pathophysiology of aPL antibodies has attracted considerable interest in the last few years. Several mechanisms have been proposed. There is now evidence that aPL may induce thrombosis by interfering with the anticoagulant properties of annexin A5. The displacement of annexin V by patients’ antibodies may result in the acceleration of procoagulant activity, thereby promoting thrombogenesis in APLAS. Aaron Tomer, J Lab Clin Med 2002;139:147-54.

13. Anti-Phospholipid Antibody Syndrome Characteristics of Patients Total 154 Men 31 (20%) Women 123 (80%) Gender Mean 41.3±12.7 ( Range 20-69) Age (years) Total 154 APS* 66 (42.9%) SLE 39 (25.3%) Normal 49 (31.8%) Clinical Diagnosis Arterial thrombosis 33 (39%) Venous thrombosis 14 (17%) Pregnancy complications 29 (35%) Thrombocytopenia 7 (8%) Clinical Presentation * of 66 patient, 44 (66.6%) have a primary disease, …22 (33.30) have APS associated with SLE

14. Anti-Phospholipid Antibody Syndrome Anti-Phospholipid Antibody Syndrome Flow Cytometric Annexin A5 Competition Assay Binding of Annexin A5

15. Anti-Phospholipid Antibody Syndrome Anti-Phospholipid Antibody Syndrome Comperison of FCA with ACL Assay TotalACL +ACL -Assay 691455 (73%) FCA - 8565 (82%) 20* FCA + 1547975 Total * 12 LAC + ; 9 APS±SLE, 10 SLE, 1NL

16. Anti-PhospholipidAntibody Syndrome Anti-PhospholipidAntibody Syndrome Comperison of FCA with LAC Assay TotalLAC +LAC -Assay 681157 (72%) FCA - 8664 (85%) 22* FCA + 1546569 Total * 12 ACL + ; 11 APS± SLE, 10 SLE, 1NL

17. Anti-PhospholipidAntibody Syndrome Anti-PhospholipidAntibody Syndrome Comperison of FCA with aPL-ab Assays TotalaPL +aPL -Assay 691851 (86%) FCA - 8577 (81%) 8*8* FCA + 1549559 Total

18. FCA (%) LAC (%) aCL (%) Number of Patients Clinical Presentation 97.093.9 81.8*33Arterial thrombosis 71.485.778.614Deep vein thrombosis 10058.679.329 Pregnancy vascular complications 100 7Thrombocytopenia * Percent positive Antiphospholipid Antibody Syndrome Sensitivity of the laboratory assays according to the clinical manifestation of APS

19. Anti-Phospholipid Antibody Syndrome Anti-Phospholipid Antibody Syndrome ROC Plot Analysis for Diagnosis of APS Area under the curve (AUC) Labratory test.990FCM.714ACLIgM.899ACLIgG.890LACRVVT.849LACPTT FCA Sensitivity: 95 Specificity: 97

20. Conclusions  The flow cytometric annexin A5 competition assay: Combines sensitivity of both ACL and LAC assays Highly sensitive for arterial events High specificity Might have significant positive predictive value for arterial thrombosis and pregnancy vascular complications  Competition of the APL-abs with Annexin A5 on the platelet anionic-phospholipids may: Interfere with Annexin A5 anticoagulant effect Results in accelerated prothrombotic activity in APS Anti-phospholipid Antibody Syndrome

21. Mahmoud Abu-Shakra Rheumatology Clinic Soroka University Medical Ctr, Israel Boris Shenkman Coangulation Laboratory Sheba Medical Center, Israel Shira Bar-Lev Ben-Gurion University, Israel Contributors